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2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) Atropisomers Interact Enantioselectively with Hepatic Microsomal Cytochrome P450 Enzymes Izabela Kania-Korwel,† Eugene G. Hrycay,‡ Stelvio M. Bandiera,‡ and Hans-Joachim Lehmler*,† Department of Occupational and EnVironmental Health, UniVersity of Iowa, 100 Oakdale Campus #124 IREH, Iowa City, Iowa 52242 and Faculty of Pharmaceutical Sciences, UniVersity of British Columbia, 2146 East Mall, VancouVer, British Columbia, Canada V6T 1Z3 ReceiVed February 13, 2008
2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) is a chiral and highly neurotoxic PCB congener of environmental relevance. (+)-PCB 136 was previously shown to be enriched in tissues from mice treated with racemic PCB 136. We investigated the spectral interactions of (+)-, (-)-, and (()-PCB 136 with mouse and rat hepatic microsomal cytochrome P450 (P450) enzymes to test the hypothesis that enantioselective binding to specific P450 enzymes causes the enrichment of (+)-PCB 136 in ViVo. Hepatic microsomes prepared from C57BL/6 mice or Long Evans rats treated with β-naphthoflavone or 3-methylcholanthrene, phenobarbital, and dexamethasone (prototypical inducers of CYP1A, CYP2B, and CYP3A, respectively) were used to determine first, whether the (+)-PCB 136 atropisomer binds to hepatic microsomal P450 enzymes to a greater extent than does the (-)-PCB 136 atropisomer and second, whether P450 enzymes of one subfamily bind the two PCB 136 atropisomers more efficiently than do P450 enzymes of other subfamilies. Increasing concentrations of (+)-, (-)-, or (()-PCB 136 were added to hepatic microsomes, and the difference spectrum and maximal absorbance change, a measure of PCB binding to P450 enzymes, were measured. A significantly larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four hepatic microsomal preparations in mice and rats, indicating that (+)-PCB 136 interacted with microsomal P450 enzymes to a greater degree than did (-)-PCB 136. In addition, binding of the PCB 136 atropisomers was greatest in microsomes from PBtreated mice and rats and was inhibited by CYP2B antibodies, indicating the involvement of CYP2B enzymes. Together, these results suggest preferential binding of (+)-PCB 136 to P450 enzymes (such as CYP2B and CYP3A) in hepatic microsomes, an observation that may explain the enantioselective enrichment of the (+)-PCB 136 atropisomer in tissues of mice. Introduction 1
Polychlorinated biphenyls (PCBs ) are a group of 209 semivolatile, persistent organic pollutants with the general structure C12HnCl10-n (1). They were manufactured by batch chlorination of biphenyl and are still used in enclosed systems, such as capacitors and transformers. Individual PCB congeners have physicochemical properties that are determined by their degree of chlorination and their substitution pattern and differ by several orders of magnitude (2). PCBs are highly lipophilic, semivolatile compounds and have excellent oral bioavailability. These properties led to worldwide contamination by PCBs and have resulted in considerable human exposure, especially via the diet. Individual PCB congeners cause adverse human health effects by different modes of action depending on their threedimensional structure. Recent epidemiological studies have * To whom correspondence should be addressed. Tel: 319-335-4414. Fax: 319-335-4290. E-mail:
[email protected]. † University of Iowa. ‡ University of British Columbia. 1 Abbreviations: ∆A, absorbance change; ∆Amax, maximal absorbance change; CO, corn oil; CYP, designates an individual cytochrome P450 enzyme; DEX, dexamethasone; DMSO, dimethyl sulfoxide; IgG, immunoglobulin G; Ksapp, apparent spectral dissociation constant; NF, β-naphthoflavone; MC, 3-methylcholanthrene; P450, cytochrome P450; PB, phenobarbital; PCBs, polychlorinated biphenyls; PCB 84, 2,2′,3,3′,6pentachlorobiphenyl; PCB 136, 2,2′,3,3′,6,6′-hexachlorobiphenyl.
shown that exposure to PCB mixtures is linked to adverse developmental outcomes and neurotoxicity (3, 4). On the basis of structure-activity studies, only PCB congeners with multiple ortho chlorine substituents are neurotoxic in Vitro and in ViVo. Several highly neurotoxic PCB congeners with three or four ortho chlorine substituents exist as two stable rotational isomers (or atropisomers) and are chiral because of an unsymmetrical substitution pattern in both phenyl rings (5). Chiral PCB congeners are important constituents of both technical and environmental PCB mixtures. For example, the total concentration of chiral PCB congeners in Aroclors 1242 and Aroclor 1260 is 6% and 30% by weight, respectively (6). There is evidence that PCB atropisomers differ in their biological activities. Early studies have shown that pure PCB atropisomers differ in their potential to induce hepatic enzymes, such as cytochrome P450 (P450) enzymes and others (7). More recent studies have shown that PCB 84 atropisomers differ in their effect on protein kinase C translocation in cerebellar granule cells, a biological measure associated with neurotoxicity (8). There is also evidence that PCB 136 atropisomers enantioselectively activate ryanodine receptors RyR1 and RyR2 (9). Pure PCB atropisomers not only cause adverse biological effects with different potencies but also are subject to enantioselective disposition processes due to enantioselective interactions with proteins. Several environmental studies have shown
10.1021/tx800059j CCC: $40.75 2008 American Chemical Society Published on Web 05/22/2008
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Figure 2. Type I difference spectra elicited by the addition of (+)PCB 136 (A) or (-)-PCB 136 (B) to hepatic microsomes from PBtreated mice (representative spectra of 3 determinations). Indicated concentrations of PCB 136 (in DMSO) were added in 10 µL quantities separately to 1 mL sample cuvettes containing microsomes (4 nmol CYP/mL) in 0.1 M potassium phosphate buffer (pH 7.5). Reference cuvettes received 10 µL of DMSO. Difference spectra were recorded separately for each PCB concentration as described in Experimental Procedures. Spectra were scanned and digitized using Engauge Digitizer software (http://digitizer.sourceforge.net/).
Figure 1. Separation of PCB 136 atropisomers using chiral gas chromatography. Representative chromatogram illustrating the enrichment of (+)-PCB 136 in the liver of a PB-pretreated mouse (22) (A) as well as chromatograms of pure (+)-PCB 136 atropisomer (B), pure (-)-PCB 136 atropisomer (C), and racemic PCB 136 standard (D) used in the spectral binding studies. The PCB atropisomers were separated using two serially connected Nucleodex P-PM columns as described in Experimental Procedures.
enantiomeric enrichment of chiral PCBs in fish, marine mammals, and in humans (5). Enrichment of (+)-PCB 136 has also been reported in Arctic char (10) and eggs from predatory birds (11), whereas (-)-PCB 136 was enriched in rainbow trout (12, 13), wolverine (14), and Arctic fox (14). In contrast, neither atropisomer was enriched in the freshwater invertebrate Mysis relicta (15) or in liver samples from bowhead whales (16) or whales from the Mediterranean Sea (17, 18). Similarly, laboratory studies investigating either individual PCB congeners or technical PCB mixtures have reported an enantiomeric enrichment in blood and tissues from mice (6, 19, 20) and rats (21). The extent of the enantiomeric enrichment depends on the route of administration (19) and the PCB dose (6). Moreover, increased enrichment of (+)-PCB 136 was observed in the liver of mice that had been pretreated with β-naphthoflavone (NF), phenobarbital (PB), or dexamethasone (DEX) compared to corn oil (CO) (22) (Figure 1A).
The enantioselective processes responsible for the enantiomeric enrichment of PCBs are poorly understood, but several studies suggested enantioselective biotransformation by P450 enzymes or binding to transport proteins (23–25). It has been shown that PCB 136 is rapidly metabolized by P450 enzymes in rodents (26–29). Thus, enantiomeric enrichment of (+)-PCB 136 in rodents may be due to the more rapid metabolism of (-)-PCB 136 by hepatic P450 enzymes. P450 enzymes differ in their ability to metabolize individual PCB congeners (30). The ability of PCBs to bind to P450 enzymes is influenced by the position of the chlorine substituents, which determines the degree of coplanarity of the phenyl rings. For instance, PCB congeners with four ortho chlorine substituents are poor substrates for hepatic CYP1A enzymes but have been shown to bind to microsomal CYP2B enzymes (30). However, it is not known if PCB atropisomers interact with P450 enzymes in an enantioselective manner. To investigate the hypothesis that enantioselective binding to specific P450 enzymes leads to enrichment of (+)-PCB 136 in ViVo, we measured the spectral binding properties of PCB 136 atropisomers to hepatic microsomes prepared from mice and rats that had been pretreated with prototypical inducers of CYP1A, CYP2B, and CYP3A enzymes (NF or 3-methylcholanthrene (MC) for CYP1A, PB for CYP2B, and DEX for CYP3A).
Experimental Procedures Caution: PCBs are hazardous and should be handled carefully. Materials. A detailed description of the batch of (()-PCB 136 (racemic mixture) used in this study was reported previously (31). Briefly, (()-PCB 136 was synthesized by the Ullmann coupling reaction of 2,3,6-trichloro-1-iodobenzene and purified using charcoal to remove trace amounts of halogenated dibenzofurans. PCB 136
Spectral Binding of PCB 136 Atropisomers to P450
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Figure 3. Representative plots of the absorbance changes elicited by increasing concentrations of (+)-, (-)-, or (()-PCB 136 with mouse hepatic microsomes (3 experiments for PB and DEX; single experiment for NF and CO). Increasing amounts of PCB were added separately to 1 mL sample cuvettes containing hepatic microsomes, at 4 nmol P450/mL, prepared from NF- (A), PB- (B), DEX- (C), and CO-treated (D) mice. Difference spectra were recorded and ∆A values calculated as described in Experimental Procedures.
Table 1. Spectral Constants and Relative Binding Efficiencies for PCB 136 Atropisomers with Mouse Hepatic Microsomal P450 Enzymesa (+)-PCB 136
(-)-PCB 136
(()-PCB 136
Hepatic Microsomes from NF-Treated Miceb ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M)
Spectral parameter
0.011 9.2 1200
0.005 2.9 1900
0.008 4.2 2000
Hepatic Microsomes from PB-Treated Micec ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M)
0.028 ( 0.002 8.2 ( 0.70 3500 ( 450
0.020 ( 0.002 5.6 ( 1.0 3800 ( 480
0.027 ( 0.001 7.0 ( 0.60 3900 ( 430
Hepatic Microsomes from DEX-Treated Micec ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M)
0.026 ( 0.001 24 ( 5.4 1200 ( 240
0.017 ( 0.001 7.6 ( 1.4 2400 ( 450
0.020 ( 0.001 12 ( 2.8 1800 ( 380
Hepatic Microsomes from CO-Treated Miceb ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M)
0.012 9.7 1200
0.010 21 450
0.012 10 1200
a Maximal absorbance change (∆Amax), apparent spectral dissociation constant (Ksapp), and relative binding efficiency (∆Amax/Ksapp) values were obtained for hepatic microsomes prepared from NF, PB, DEX, and CO-treated female mice as described in Experimental Procedures. b Single measurement. c Mean ( SE of three determinations.
atropisomers were separated by reversed phase-high pressure liquid chromatography on two serially connected 4.6 × 250 mm Nucleodex β-PM columns (Macherey-Nagel, Du¨ren, Germany), as described by Haglund (32). As shown in Figure 1, the enantiomeric purity of the PCB 136 atropisomers was determined using a HP6890 gas chromatograph equipped with a 63Ni µ-ECD detector and a Chirasil-Dex column (β-cyclodextrin chemically bonded with dimethylpolysiloxane, 25 m × 0.25 mm ID × 0.25 µm film thickness, Varian, Palo Alto, CA) (6, 19, 20). The enantiomeric fractions (EF) of (-)-PCB 136 and (+)-PCB 136 were 0.01 and 1.00, respectively (EF ) peak area (+)-PCB 136/[peak area (+)PCB 136 + peak area (-)-PCB 136] according to Harner et al. (33)). Alkaline phosphatase-conjugated goat antirabbit F[ab′]2 immunoglobulin G (IgG) was purchased from BioSource International (Camarillo, CA). Other chemicals were obtained from commercially available sources.
Animal Treatment. Experiments involving mice were approved by the Institutional Animal Care and Use Committee at the University of Iowa. Female 8-week old C57BL/6 mice (n ) 104, average weight ) 18.2 ( 1.1 g) were obtained from Harlan (Indianapolis, IN). Mice were allowed to acclimatize for one week before being randomly assigned to four treatment groups. Mice received intraperitoneal injections of NF in CO (n ) 27, 27 mg/ kg/day), PB in saline (n ) 25, 102 mg/kg/day), DEX in CO (n ) 25, 50 mg/kg/day), or CO alone (n ) 27, 10 mL/kg/day) for 3 consecutive days (22). Mice were killed by CO2 asphyxiation followed by cervical dislocation 24 h after the last treatment. Livers were excised and immediately placed in ice-cold 0.05 M Tris-HCl buffer (pH 7.5) containing 0.15 M KCl. Rats were housed at the University of British Columbia (Vancouver, British Columbia, Canada) and were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Adult male Long Evans rats were purchased from
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Figure 4. Representative plots of absorbance changes elicited by increasing concentrations of (+)-, (-)-, or (()-PCB 136 with rat hepatic microsomes (3 separate experiments for PB, DEX, and CO; single experiment for MC). Increasing amounts of PCB were added separately to 1 mL sample cuvettes containing hepatic microsomes at 4 nmol P450/mL prepared from MC- (A), PB- (B), DEX- (C), and CO-treated (D) rats. Difference spectra were recorded and ∆A values calculated as described in Experimental Procedures.
Table 2. Spectral Constants and Relative Binding Efficiencies for PCB 136 Atropisomers with Rat Hepatic Microsomal P450 Enzymesa Spectral parameter
(+)-PCB 136
(-)-PCB 136
(()-PCB 136
Hepatic Microsomes from MC-Treated Rats ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M)
0.012 13 920
0.010 6.9 1500
0.011 8.8 1300
Hepatic Microsomes from PB-Treated Ratsc ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M)
0.060 ( 0.009 2.2 ( 0.47 28000 ( 2800
0.053 ( 0.006 3.8 ( 0.76 14000 ( 1500
0.058 ( 0.007 2.6 ( 0.48 23000 ( 2100
0.036 ( 0.005 12 ( 2.4 3100 ( 240
0.024 ( 0.003 16 ( 3.2 1600 ( 130
0.032 ( 0.004 12 ( 2.8 2800 ( 360
0.046 ( 0.001 9.2 ( 1.6 5300 ( 920
0.042 ( 0.001 10 ( 1.1 4000 ( 420
0.044 ( 0.001 8.8 ( 1.2 5300 ( 900
b
Hepatic Microsomes from DEX-Treated Ratsc ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M) Hepatic Microsomes from CO-Treated Ratsc ∆Amax (absorbance unit/nmol P450) Ksapp (µM) ∆Amax/Ksapp (absorbance unit/nmol P450/M)
a Maximal absorbance change (∆Amax), apparent spectral dissociation constant (Ksapp), and relative binding efficiency (∆Amax/Ksapp) values were obtained for hepatic microsomes prepared from MC, PB, DEX, and CO-treated male rats as described in Experimental Procedures. b Single measurement. c Mean ( SE of three determinations.
Charles River Laboratories (Montreal, Quebec, Canada) and were randomly assigned to four treatment groups with six animals per group. Rats received intraperitoneal injections of MC in CO (n ) 6, 25 mg/kg/day), PB in saline (n ) 6, 80 mg/kg/day), DEX in CO (n ) 6, 100 mg/kg/day), or CO alone (n ) 6, 2 mL/kg/day) for 3 consecutive days. Rats were killed by decapitation 24 h after the last treatment. Livers were excised and immediately placed in icecold 0.05 M Tris-HCl buffer (pH 7.5) containing 0.15 M KCl. Preparation of Hepatic Microsomes. Mouse and rat hepatic microsomes were prepared by differential ultracentrifugation as described previously (34). Liver homogenates were prepared from individual livers and were then pooled for mice or rats from each treatment group. The pooled homogenates were spun at 9,000g for 20 min, and the resulting supernatants were subjected to two consecutive centrifugations at 100,000g for 60 min. The microsomal pellets were resuspended in 0.25 M sucrose, and 1 mL aliquots were stored at -80 °C. Protein concentrations were measured by
the method of Lowry et al. (35) using bovine serum albumin as a standard. Total P450 concentrations were determined by the method of Omura and Sato (36) using an extinction coefficient of 91 mM-1 cm-1. Binding of PCB 136 to Hepatic Microsomal P450. The binding of (+)-, (-)-, and (()-PCB 136 to hepatic microsomal P450 enzymes was studied by recording the type I difference spectra at room temperature using an Aminco DW-2 dual wavelength/split beam spectrophotometer as described previously (37). Briefly, 0.99 mL of a hepatic microsomal preparation (diluted to 4 nmol total P450/mL in 0.1 M potassium phosphate buffer, pH 7.5) was added to both sample and reference cuvettes. After the baseline was recorded, 10 µL of (+)-, (-)-, or (()-PCB 136 dissolved in dimethyl sulfoxide (DMSO) was added to the sample cuvette, while 10 µL of DMSO was added to the reference cuvette to offset any contribution made by DMSO to the difference spectrum. DMSO alone (at 10 µL/mL) did not produce a type I difference spectrum.
Spectral Binding of PCB 136 Atropisomers to P450
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Table 3. Relative P450 Protein Levels in Mouse Hepatic Microsomesa immunoreactive P450 protein levels treatment
CYP1A2b
CYP2Bc
CYP3Ad
NF PB DEX CO
9.5 ( 0.5 2.1 ( 0.2 0.4 ( 0.1 1.0 ( 0.1
1.1 ( 0.1 15.2 ( 1.1 14.9 ( 1.1 1.0 ( 0.1
1.0 ( 0.1 5.3 ( 0.3 25.9 ( 1.0 1.0 ( 0.2
a P450 protein levels are expressed relative to those of CO-treated mice set at 1.00. The amount of immunoreactive P450 protein was calculated in terms of relative optical density units/mg of microsomal protein because purified or recombinant mouse P450 standards are not available. Values shown are the experimental mean ( SE of 3 or 4 determinations using pooled hepatic microsomes prepared from female mice that had been pretreated with NF, PB, DEX, or CO. Purified or recombinant rat P450 enzymes were included on the gels as reference standards. Immunoreactive P450 protein bands were detected with antibodies directed against rat P450 enzymes as described in Experimental Procedures. b Only relative CYP1A2 levels are reported. CYP1A1 was not detected in hepatic microsomes from PB-, DEX- or CO-treated mice but was detected in hepatic microsomes from NF-treated mice. A relative CYP1A1 level was not determined for NF-treated mice due to the lack of a reference point. c The CYP2B level reported here corresponds to the immunoreactive band tentatively identified as CYP2B10 by reference to the electrophoretic mobility previously reported (52). d The CYP3A level reported here corresponds to the immunoreactive band tentatively identified as CYP3A11 by reference to the electrophoretic mobility previously reported (52).
The difference spectrum was recorded with repetitive scanning between 350 and 500 nm. The absorbance change was determined as the difference between the spectral peak (λmax at approximately 390 nm) and trough (λmin at approximately 420 nm) and was expressed per nmol of P450. The apparent spectral dissociation constant (Ksapp) and maximal absorbance change (∆Amax) were calculated by nonlinear regression analysis using Enzyme Kinetics Module of Sigma-Plot (Systat Software Inc., Ver. 10, San Jose, CA). The ∆Amax/Ksapp parameter, which reflects the relative binding efficiency (38, 39), was calculated to facilitate comparisons. Immunoblot Analysis. Immunoblot analysis was used to measure protein levels of CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP2C11, and CYP3A enzymes in rat hepatic microsomes (37) and relative levels of immunoreactive CYP1A, CYP2B and CYP3A enzymes in mouse hepatic microsomes. Microsomal proteins were resolved by SDS-PAGE, then transferred onto nitrocellulose membranes, and probed with rabbit antirat CYP1A2 polyspecific antiserum (1:1000 dilution), anti-CYP2B1 polyspecific IgG (2 µg/ mL), anti-CYP2C11 monospecific IgG (10 µg/mL), or anti-CYP3A polyspecific IgG (5 µg/mL), at the concentrations indicated. Purified rat CYP1A1 (0.05-0.375 pmol/lane), CYP1A2 (0.025-0.25 pmol/ lane), CYP2B1 (0.031-0.25 pmol/lane), CYP2C11 (0.1-0.4 pmol/ lane), and rat recombinant CYP3A1 and CYP3A2 (0.125-0.375 pmol/lane) were applied onto the gels as calibration/reference standards in a volume of 20 µL/lane, at the concentration ranges indicated. The preparation of rat P450 enzymes and antibodies against rat P450 enzymes was described previously (37). Antibody and Serum Inhibition Studies. Serum was obtained from adult female New Zealand rabbits that had been immunized with purified, electrophoretically homogeneous CYP2B1, CY2C11, or CYP3A1. Each rabbit was immunized initially with 200 µg of one of the purified P450 enzymes emulsified in Freund’s complete adjuvant and administered subcutaneously. Three rabbits were immunized with each purified P450 enzyme. Four weeks later, the rabbits received a boost consisting of 50 µg of the P450 protein per rabbit as a 1:1 emulsion in Freund’s incomplete adjuvant administered by intramuscular injection. Thereafter, rabbits were boosted on a monthly basis by intravenous injection of the P450 protein (50 µg dissolved in phosphate-buffered saline, pH 7.4) into the ear vein, followed by collection of approximately 30 mL of blood from each rabbit 7 days after the boost. Blood from each bleeding was allowed to clot at 37 °C, and serum was removed
and stored at -20 °C. Control serum was obtained from rabbits that were not immunized. Increasing volumes (0.05-0.5 mL) of serum were added to both sample and reference cuvettes containing hepatic microsomes (2 nmol P450/mL in 0.1 M potassium phosphate buffer, pH 7.5). The volume was brought up to 0.99 mL with phosphate buffered saline, where necessary. The microsomal samples were preincubated with the serum in the cuvettes at room temperature for 5 min, followed by addition of 10 µL of (+)-, (-)-, or (()-PCB 136 (2.5 mM in DMSO) to the sample cuvette and 10 µL of DMSO to the reference cuvette. The binding spectra were then recorded as described above. Statistical Analysis. Data are presented as the experimental mean ( SE where applicable. Differences between mean values were analyzed using open-source R statistical software (ver. 2.5.1).
Results Difference Spectra Using Mouse Hepatic Microsomes. Addition of (+)-PCB 136, (-)-PCB 136, or PCB 136 racemate to hepatic microsomes prepared from NF-, PB-, DEX-, or COtreated female mice yielded type I difference spectra, with a λmax at 385-390 nm and a λmin at 416-422 nm. Representative difference spectra for (+)- and (-)-PCB 136, at various concentrations, are presented in Figure 2. The effect of increasing PCB concentration on absorbance change can be seen more clearly in Figure 3. The magnitude of absorbance change varied with the microsomal preparation used and increased with increasing PCB concentration up to a concentration of 50-100 µM. A larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four microsomal preparations at PCB concentrations greater than 25 µM. Values of maximal absorbance change (∆Amax), apparent spectral affinity (Ksapp) and relative binding efficiency (∆Amax/ Ks), were calculated for (+)-, (-)-, or (()-PCB 136 and are presented in Table 1. These spectral parameters reflect the ability of PCB 136 atropisomers to bind to hepatic microsomal P450 enzymes. Maximal absorbance change values and relative binding efficiency values for both PCB 136 atropisomers and for racemic PCB 136 were much larger with hepatic microsomes prepared from PB-treated mice than NF- or CO-treated mice, suggesting that (+)-PCB 136 and (-)-PCB 136 are interacting to a greater extent with one or more P450 enzymes present in the liver of PB-treated mice than with the P450 enzymes found in the liver of NF- or CO-treated mice. Moreover, ∆Amax values were greater for (+)-PCB 136 than (-)-PCB 136 for all four microsomal preparations. However, there was no clear rank order in the relative binding efficiency of PCB 136 atropisomers within the four microsomal preparations. For instance, the relative binding efficiency of (-)-PCB 136 with hepatic microsomes from NF- or DEX-treated mice was greater than that obtained for (+)-PCB 136 and was smaller than that obtained for (+)-PCB 136 with hepatic microsomes from COtreated mice. Difference Spectra Using Rat Hepatic Microsomes. To determine if preferential binding of PCB 136 with hepatic microsomes from PB-treated mice and the enantiomeric differences observed are characteristic of other species, the spectral binding studies were repeated with rat hepatic microsomes. Similar results but larger absorbance changes were obtained when rat hepatic microsomes were used (Figure 4). As shown in Table 2, the spectral parameters determined using rat hepatic microsomes and (+)-, (-)-, or (()-PCB 136 were comparable to those observed using mouse hepatic microsomes. The largest ∆Amax values were observed for PCB 136 atropisomers with microsomes from PB-treated rats, and the smallest ∆Amax values were observed for PCB 136 atropi-
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Table 4. P450 Protein Levels in Rat Hepatic Microsomesa P450 protein levels (pmol/nmol total P450) treatment MC PB DEX CO
CYP1A1
CYP1A2
CYP2B1
CYP2B2
370 ( 12 (9) MC in rats. The same rank order of binding to rat hepatic microsomes has been reported for PCB 54, a tetrachlorobiphenyl that, like PCB 136, has four ortho chlorine substituents (37). Similarly, the relative binding efficiency of (+)- and (-)-PCB 136 was much larger with hepatic microsomes from mice and rats that had been pretreated with PB compared to the other inducers. Overall, the spectral constants
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and binding efficiencies suggest that both PCB 136 atropisomers preferentially bind to CYP2B enzymes, which are the predominant P450 subfamily in microsomes prepared from PB- and, to a lesser extent, DEX-treated animals. Inhibition studies were performed with selected rabbit antirat P450 serum samples to verify that PCB 136 atropisomers indeed bind to rat P450 enzymes. Analogous studies with mouse microsomes were not performed because of the unavailability of antibodies specific for mouse P450 enzymes. We focused on CYP2B and CYP3A enzymes in the inhibition studies because multi ortho-substituted PCBs (such as PCB 54) are known to interact with both P450 subfamilies (37) and because PCB 136 is known to be metabolized by CYP2B enzymes (48–50). Furthermore, experiments were performed only with microsomes from PB- and DEX-treated rats because, in agreement with previous results (37, 40–42), CYP2B is the major P450 enzyme in microsomes from PB-treated rats, and CYP3A predominates in microsomes from DEX-treated rats (Table 4). The antibody inhibition studies confirmed that PCB 136 atropisomers bind preferentially to CYP2B and CYP3A enzymes in microsomes from both PB- and DEX-treated rats. The observation that PCB 136 atropisomers bind efficiently to CYP2B enzymes is not surprising because, as mentioned above, PCB 136 is readily metabolized by this subfamily of P450 enzymes (48–50). The finding that PB induction leads to increased enantiomeric enrichment of (+)-PCB 136 in mice (22) further supports a role for CYP2B enzymes in the disposition of PCB 136 atropisomers. However, it is currently unclear if PCB 136 atropisomers are also metabolized by CYP3A enzymes, a question that warrants further investigation with purified P450 enzymes. The most intriguing finding of our study was that (+)-PCB 136 appeared to interact to a greater degree than (-)-PCB 136 with microsomes from mice and rats, independent of the pretreatment, and is based on the absorbance changes observed with increasing PCB 136 atropisomer concentrations (see Figures 3 and 4) and the maximal absorbance changes (∆Amax) reported in Tables 1 and 2. This finding suggests preferential binding of (+)-PCB 136 to P450 enzymes such as CYP2B and CYP3A present in the microsomal preparations. If (+)-PCB 136 is preferentially metabolized by CYP2B and possibly CYP3A enzymes, it is difficult to explain why (+)-PCB 136 is enriched in mouse tissues. However, a slightly analogous situation exists for a related group of compounds. Diliberto et al. (51) found high levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin and other laterally chlorinated dibenzo-p-dioxins and dibenzofurans in the liver of wild-type mice but not in the liver of CYP1A2 knockout mice. On the basis of the results, these investigators proposed that hepatic sequestration of laterally chlorinated dibenzo-pdioxins and dibenzofurans is due to induction and binding of CYP1A2. Analogous to the work by Diliberto et al. (51), the present study suggests two mechanisms that may be responsible for the enantiomeric enrichment of (+)-PCB 136 in tissues of these experimental animals. The first mechanism involves hepatic sequestration of (+)-PCB 136 due to enantioselective binding of the atropisomer to P450 enzymes. Similarly, the sequestration of dioxin-like PCB congeners is thought to be the result of binding to CYP1A2 enzymes (51). The second possible mechanism also involves preferential binding of the (+)-PCB 136 atropisomer to CYP2B enzymes, which results in a more rapid metabolism of the (-)-PCB 136 atropisomer due to the latter’s weaker binding to CYP2B enzymes. Further in Vitro studies with purified P450 enzymes are needed to determine the binding
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efficiencies and rates of metabolism of both PCB 136 atropisomers, and these studies could be extended to other chiral PCB congeners to establish whether an inverse relationship exists between the binding of PCB substrates to, and their metabolism by, P450 enzymes. Acknowledgment. This work was supported by the following grants ES05605, ES013661, and ES012475 from the National Institute of Environmental Health Sciences, NIH. S.M.B. wishes to acknowledge the Natural Sciences and Engineering Council of Canada (RGPIN 138733-01) for their financial support.
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